This work will involve investigations into the mechanism whereby insulin stimulates the sodium pump and membrane-bound Na ion-K ion ATPase. The principal approach will involve kinetic studies on intact muscle cells coupled with studies of the Na ion-K ion ATPase isolated from muscle as well as from brain. The possibility that the sodium pump can also transport H ion will be investigated as will the hypothesis that insulin causes the pump to efflux H ion and as a result increases intracellular pH (pHi). The flux experiments will be conducted mostly on frog skeletal muscles with the muscle from one leg serving as a paired control for the other. Many of the enzyme experiments will be conducted on Na ion-K ion ATPase isolated from rat brain. The first objective will be to test the hypothesis that insulin stimulation of the sodium pump is due to an increase in the association constant for Na ion of the carrier sites facing the inner side of the membrane. This hypothesis will be tested by comparing the effect of insulin upon Na22 efflux from frog muscles at low concentrations of intracellular Na ion to the effect at concentrations sufficiently elevated to saturate the sites. A second objective will be to measure changes in intracellular pH (pHi) and to determine if an active efflux of H ion exists which is mediated by the sodium pump. A third, and closely related objective, will be to determine if insulin increases pHi. Intracellular pH will be determined by the use of DMO-C14 (C14 5,5-dimethyl 1-2, 4-oxazolidinedione) as well as by pH sensitive glass microelectrodes. A fourth objective will be to study the effect of Na ion, K ion, and other allosteric effectors upon the response of the Na ion-K ion ATPase system to insulin. Any advances in understanding which may come from this work will very likely increase our insight into the molecular and cellular action of insulin as well as into the reason for the frequent failure of long-term diabetic therapy. BIBLIOGRAPHIC REFERENCES: W. A. Gavryck, R. D. Moore and R.C. Thompson. 1975. Effect of insulin upon membrane-bound (Na plus K ions)-ATPase extracted from frog skeletal muscle J. Physiol. 252:43-58. S.Y. Chan and R.D. Moore. February 1976. Effect of sanguinarine upon sodium efflux from frog skeletal muscle. Biophysical Journal Abstracts 16:28a.